The missing link to synthetic agar

Agar consists of many linked subunits of the sugar galactose. Agar is used as a gelling agent in food production and also in medical and microbial research. Agar is particularly popular as a vegan substitute for gelatine in food production. Agar is produced from the cell walls of red algae by extraction with hot water. These red algae are harvested worldwide on the coastal areas of all five continents. Depending on the origin and type of the harvested red algae, the quality and properties of the agar produced can vary greatly. The amount of red algae as a natural resource is limited and the harvest can only barely meet the annual demand for agar. It is therefore desirable to develop an alternative allowing for the biotechnological production of agar. To do this, one has to understand how agar is made in red algae and know every step of agar biosynthesis. Most of the steps in this synthesis route are already known. However, a decisive step on this path is completely unknown. It is the step in which the galactose units are linked into a long chain to form an agar molecule. This linkage is catalyzed by an enzyme called galactosyl transferase. The aim of this project is to identify this galactosyl transferase and to find out more about its localization and activity in red algae. Computer-aided bioinformatics and experimental biochemical methods are used for identification. Bioinformatics is used to search the genome of the agar-producing red algae. In order to detect the enzymes identified by bioinformatics or to find additional enzymes, the so-called Golgi apparatus of the red algae will be isolated and purified using biochemical working methods. The galactosyl transferases are, according to general scientific opinion, associated with or even contained in the Golgi apparatus. The final test of the galactosyl transferase activity is carried out through the overproduction of the identified enzymes in yeast. The enzymes produced in this way can be isolated from the yeast and characterized for their activity. As soon as one or more galactosyl transferases are identified and characterized, this opens up the possibility of producing agar that does not solely rely on the natural and limited resource of red algae and can also be tailored to special requirements.

Abstract TAI_515_B The missing link to synthetic agar The main objective of this project was to identify candidate galactosyl transferases which are responsible for the biosynthesis of agar in red algae Agar consists of many linked subunits of the sugar galactose. Agar is used as a gelling agent in food production and also in medical and microbial research. Agar is particularly popular as a vegan substitute for gelatin in food production. Agar is produced from the cell walls of red algae by extraction with hot water. These red algae are harvested worldwide on the coastal areas of all five continents. Depending on the origin and type of the harvested red algae, the quality and properties of the agar produced can vary greatly. The amount of red algae as a natural resource is limited and the harvest can only barely meet the annual demand for agar. It is therefore desirable to develop an alternative allowing for the biotechnological production of agar. To do this, one has to understand how agar is made in red algae and know every step of agar biosynthesis. Most of the steps in this synthesis route are already known. However, a decisive step on this path is completely unknown. It is the step in which the galactose units are linked into a long chain to form an agar molecule. This linkage is catalyzed by an enzyme called galactosyl transferase. The aim of this project was to identify this galactosyl transferase and to find out more about its localization and activity in red algae. Computer-aided bioinformatics and experimental biochemical methods were used for identification. Bioinformatics was used to search the genome of the agar-producing red algae species Gracilaria chorda. The combination of bioinformatics and protein identification based on the isolation of the Golgi apparatus from Gracilaria chorda enabled the identification of 20 candidate genes, out of which three were identified that were both likely to be galactosyl transferases and localize to the Golgi apparatus. These three candidates were selected for expression in the yeast Pichia pastoris, to assess their potential to produce agarose chains. Furthermore, the same analysis revealed the presence of 4 candidate genes coding for special galactose transporter proteins which mediate the transport of activated galactose to the Golgi apparatus to provide the substrate for agar synthesis to the correct localization. From these experiments we conclude that more than one galactosyl transferase is playing a central role in the production of agar. Presumably, two or more galactosyl transferases polymerize the galactose chain, which must then be sulphated, and then de-sulphated, in order to produce true gelling agar. Once the candidate enzymes have been narrowed down, it should be possible to produce agar in yeast.